Cholera, which is caused by ingestion of the Gram-negative pathogen Vibrio cholerae, persists worldwide and affects many thousands of people each year, most of whom do not have access to clean water and sanitary conditions. This disease, characterized by massive water loss and dehydration due to diarrhea, can lead to death in less than 24 hours if rehydration therapy is not initiated immediately. V. cholerae colonizes the small intestine via attachments made by the toxin co-regulated pilus (TCP) and then secretes cholera toxin from this location. Beyond cholera toxin and TCP, V. cholerae possesses many additional virulence factors, but their contributions to pathogenesis and their interactions with the innate immune system are not well understood. A genetically tractable model host along with a genetic approach to the pathogen would elucidate the host pathogen interaction in an unprecedented way. Previous work in the Watnick laboratory has established Drosophila melanogaster as a model organism for V. cholerae intestinal infection. Here we propose to use both targeted and random screens of V. cholerae mutants in a simple Drosophila survival assay in order to identify bacterial genes that contribute to virulence. We will identify loci involved in colonization, survival within the intestinal lumen, and invasion of the intestinal epithelium. Most importantly, use of the Drosophila system facilitates genetic dissection of the host response, so that the host-pathogen interaction can be studied from both perspectives. The results obtained from experiments in Drosophila may have implications for V. cholerae interactions with mammalian hosts because aspects of innate immune pathways are extensively conserved between flies and mammals. Therefore, our experimental results will be validated in a mammalian model of disease. A germ-free mouse model of colonization is being initiated in the Watnick laboratory, and competition colonization assays will be performed on mutants impaired for colonization in the fly. PUBLIC HEALTH RELEVANCE: V. cholerae causes deadly epidemic disease in developing countries. As of yet, no effective vaccine has been developed. By investigating V. cholerae infection in the fly, we hope to discover additional bacterial factors that contribute to mammalian disease. Ultimately, these results may suggest novel vaccine targets, as well as ways to modulate the immune response and interfere with colonization during human infection. In addition, arthropods in the marine environment, such as copepods, may also serve as a reservoir for this bacterium. Studies of Drosophila as model arthropods may provide insight into persistence of V. cholerae in the environment and suggest ways to prevent or limit pandemic disease.